IDENTIFICATION OF AN RNA-DEPENDENT ATPASE ACTIVITY IN MAMMALIAN U5 SNRNPS

Nuclear pre-mRNA splicing requires ATP at several steps from spliceoso me assembly to product release. Here, we demonstrate that an integral component of the 20S U5 snRNP is an RNA-dependent ATPase. The ATPase a ctivity of 20S US and 25S [U4/U6,U5] snRNPs purified by glycerol gradi ent centrifugation is strongly stimulated by homopolymeric RNA but not ssDNA, Purified 12S U1 and U2 snRNPs do not exhibit ATPase activity. Moreover the US-associated NTPase specifically hydrolyzes ATP and dATP The additional purification of 20S U5 snRNPs by Mono Q chromatography does not affect the efficiency of ATP hydrolysis. Both US and tri-snR NPs bind ATP stoichiometrically in an RNA-independent manner. A candid ate ATPase was identified by UV-irradiation of purified snRNPs with ra diolabeled ATP. In the presence of homopolymeric RNA, the 200 kDa US-s pecific protein is the major crosslinked protein, even in Mono Q-purif ied U5 snRNPs. The correlation between RNA-dependent ATPase activity i n the U5 snRNP and the RNA-dependent onset of this crosslink strongly suggests that the 200 kDa protein is an RNA-dependent ATPase. Furtherm ore, both the formation of the crosslink and ATPase activity appear wi th a similar substrate specificity for ATP.